Spodium Bonds Involving Methylmercury and Ethylmercury in Proteins: Insights from X-ray Analysis and Computations

In this study, the stability, directionality, and physical nature of Spodium bonds (SpBs, an attractive noncovalent force involving elements from group 12 and Lewis bases) between methylmercury (MeHg) and ethylmercury (EtHg) and amino acids (AAs) have been analyzed from both a structural (X-ray analysis) and theoretical (RI-MP2/def2-TZVP level of theory) point of view. More in detail, an inspection of the Protein Data Bank (PDB) reported evidence of noncovalent contacts between MeHg and EtHg molecules and electron-rich atoms (e.g., O atoms belonging to the protein backbone and S atoms from MET residues or the π-systems of aromatic AAs such as TYR or TRP). These results were rationalized through a computational study using MeHg coordinated to a thiolate group as a theoretical model and several neutral and charged electron-rich molecules (e.g., benzene, formamide, or chloride). The physical nature of the interaction was analyzed from electrostatics and orbital perspectives by performing molecular electrostatic potential (MEP) and natural bonding orbital (NBO) analyses. Lastly, the noncovalent interactions plot (NCIplot) technique was used to provide a qualitative view of the strength of the Hg SpBs and compare them to other ancillary interactions present in these systems as well as to shed light on the extension of the interaction in real space. We believe that the results derived from our study will be useful to those scientists devoted to protein engineering and bioinorganic chemistry as well as to expanding the current knowledge of SpBs among the chemical biology community.


■ INTRODUCTION
−5 This leads to a bioaccumulation and biomagnification of MeHg through the food chain, resulting in its increased concentration in higher trophic levels, including fish and seafood, which are common dietary sources for humans. 6,7MeHg exhibits particular chemical features that facilitate crossing biological barriers, such as the blood−brain barrier, 8−11 thus exerting a broad spectrum of toxicological effects on the nervous system, 12 which ultimately lead to cognitive deficits, developmental delays, and neurological disorders.−15 Beyond its neurotoxic effects, MeHg can also impact other organ systems, such as kidney function, 16 interfere with the cardiovascular system, 17 disrupt the endocrine system, 18 and weaken the immune response. 19−22 From a molecular biology perspective, MeHg acts as a disruptive agent of noncovalent interactions crucial for protein folding, stability, and function. 23,24It can bind to thiolate groups in cysteine residues, leading to conformational changes and the misfolding of proteins.In addition, it can affect DNA structure and function through the binding to DNA bases and phosphate backbone, thus provoking DNA damage, strand breaks, and interference with DNA replication and repair mechanisms. 25,26Hence, advancing in understanding the role of MeHg in biological systems is crucial for understanding its impact, assessing risks, and developing strategies for its mitigation.
In this regard, although the molecular anchoring of MeHg in a protein's cavity is based on the coordination to a CYS residue, the vicinal amino acids (AAs) also need to alter their disposition and disrupt their native network of noncovalent interactions (NCIs) to accommodate MeHg.The term Spodium bond (SpB) 27 was recently proposed to classify the NCIs involving group 12 of elements (Zn, Cd, and Hg) when acting as Lewis acids.The biological implications of this novel noncovalent force have been explored by some of us in the case of Zn, 28 thus expanding its structural and functional role in biology.SpBs are part of the "σ-hole chemistry", which involves NCIs from the p-block 29−33 and, more recently, from the d-block of elements, such as Wolfium (group 6), 34 Matere (group 7), 35 Osme (group 8), 36 and Regium bonds (group 11). 37,38These imply electrophilic regions located on the Lewis acid molecule (usually characterized by a positive electrostatic potential) that favorably interact with a Lewis base (e.g., a lone pair, a π-system, or an anion). 39n this study, our approach consisted on a combination of a Protein Data Bank (PDB) 40 survey and an ab initio theoretical study at the RI-MP2/def2-TZVP level of theory to analyze the NCIs responsible for the stabilization of MeHg and ethylmercury (EtHg) (another toxic Hg methylation derivative) in biological systems.To achieve that, an inspection of the PDB revealed 586 contacts involving MeHg and EtHg coordinated to CYS residues and electron-rich atoms (N, O, or S).Using theoretical models, we evaluated the stability and directionality of several selected Hg SpBs gathered from the PDB search.These results were complemented with an ab initio computational study at the RI-MP2/def2-TZVP level of theory.The noncovalent nature of the interaction was assessed using the quantum theory of atoms in molecules (QTAIM), the natural bonding orbital (NBO), and the noncovalent interactions plot (NCIplot) analyses.We believe the results reported herein (i) will assist in increasing the visibility of SpBs among the bioinorganic chemistry community and (ii) provide new insights into the NCIs responsible for the stabilization of MeHg and EtHg in proteins, which might have great impact in the fields of chemical biology and environmental chemistry.

■ METHODS
The RCSB website was accessed in May 2023 to download PDB files containing mercury (Hg), resulting in 694 files with at least one Hg atom at a resolution of up to 4 Å.These were filtered out for methyl or ethylmercury-containing structures, leaving 34 PDB files for further analysis.Our analysis focused on two main criteria.First, we identified the C−Hg−S moiety by examining the 2.5 Å vicinity around the Hg atom to determine sulfur (S) atom binding.Subsequently, we searched for Hg•••A SpBs, where A represented nitrogen (N), oxygen (O), or sulfur (S) atoms.The geometric criterion for Hg•••A distance was set between 2.5 and 4.5 Å and the minimum distance was kept at 2.5 Å to avoid the overlap between coordination and Spodium bonding interactions.The entire analysis was performed by using a custom Python code developed by us.
Computation of the SpB Energies in Selected PDB Structures.Once the PDB structures were identified, theoretical models were built containing MeHg/EtHg molecules coordinated to a thiolate group (CH 3 −Hg−SCH 3 ) and the interacting amino acid (composed by the side chain and the backbone atoms, see the Supporting Information for the Cartesian coordinates of the PDB models used).In a later stage, the H atoms from the PDB models were optimized at the PBE0 41,42 -D3 43 /def2-SVP 44 level of theory.These optimized geometries were then taken as a starting point for single-point calculations at the RI-MP2/def2-TZVP level of theory to compute the interaction energies given in Table 1.The interaction energies were corrected by using the Boys and Bernardi basis set superposition error (BSSE) counterpoise technique. 45omputation of the Spodium Bond Energies Using Fully Optimized Models (Complexes 1−17).The interaction energies In this complex, no A•••Hg BCP was found. of all complexes were computed at the RI-MP2 46 /def2-TZVP 44 level of theory (also corrected using the BSSE counterpoise technique), which is adequate for the treatment of NCIs involving neutral and charged electron donors. 47The calculations have been performed using the program TURBOMOLE version 7.0. 48by fully optimizing the geometries without imposing any restraints.Only the Cs symmetry point group was imposed during the optimization process (except for complex 14).The interaction energies were calculated using the supermolecule approximation The MEP (molecular electrostatic potential) surfaces were computed at the RI-MP2/def2-TZVP level of theory by means of the TURBOMOLE 7.0 program and were analyzed using Multiwfn software 49 and visualized using the Gaussview 5.0 program. 50The calculations for the wave function analysis 51 have been carried out at the RI-MP2/def2-TZVP level of theory also using Multiwfn software.The NBO 52 analyses was performed at the HF/def2-TZVP level of theory by means of the NBO 7.0 program. 53Lastly, the NCIplot 54 isosurfaces correspond to both favorable and unfavorable interactions, as differentiated by the sign of the second density Hessian eigenvalue and defined by the isosurface color.The color scheme is a red− yellow−green−blue scale with red for repulsive (ρ cut + ) and blue for attractive (ρ cut − ) NCI interaction density.Yellow and green surfaces correspond to weak repulsive and attractive interactions, respectively.The VMD 55 program was used in the visualization of the results from the QTAIM, NBO, and NCIplot analyses.
■ RESULTS AND DISCUSSION PDB Analysis.Out of the 34 PDB structures containing methyl or ethylmercury, the Hg atoms of 20 structures were coordinated to the cysteine sulfur atom.These 20 structures yielded 586 Spodium bond contacts that satisfied the imposed geometrical criteria.Further analysis of these contacts was conducted, as shown in Figure 1.In Figure 1a, the Hg•••A distance distribution demonstrates that the number of contacts increases with distance, as expected due to the increase in volume around the Hg atom and the corresponding higher probability of the presence of another atom.Additionally, the presence of two peaks near 4.0 and 4.25 Å suggests the existence of SpBs centered around the Hg atom.The X−Hg− A angle distribution, where X represents S or C atoms, is shown in Figure 1b.The distribution reveals a peak centered at around 110°, indicating that the perpendicular area to the X− Hg•••A plane experiences less steric crowding.These less crowded areas are known to be favorable for the formation of noncovalent interactions, as denoted in the radial distribution plot depicted in Figure 1c.Concretely, the plot reveals two  In order to rationalize the nature of these interactions from an electrostatics point of view, we also computed the electrostatic potential surfaces of the alkyl Hg derivatives (Figure 2d), showing an electropositive belt around the Hg atoms with a similar electrostatic potential value (+18.2 and In this complex, no BCP was found.b Distance and angle measured from the ring centroid.+16.3 kcal/mol for MeHg and EtHg, respectively) in line with that obtained for other linear transition-metal coordination complexes. 59,60It is also important to note that although the common disposition of an SpB implies a σ-hole 27,28 (involving an antibonding metal−ligand orbital), the lineal geometry observed in the Hg coordination complexes studied herein precluded the presence of a σ-hole, thus resulting in the electropositive belt observed around the Hg atom (resembling a π-hole).Furthermore, we also computed the QTAIM and NCIplot analyses of the noncovalent complexes present in these three structures, and the results show a bond critical point (BCP) connecting the lone pair donor atom (S and O) or the πsystem from the AA to the Hg atom from the MeHg/EtHg moiety, thus characterizing the SpB.In addition, ancillary CH•••CH, lone pair−π (lp−π), and hydrogen bond (HB) interactions were also present (as denoted by their corresponding BCPs and bond paths), also contributing to the binding affinities obtained.Finally, the NCIplot analyses accounted for the weak nature and extension in real space of the interaction, as is deduced from the greenish isosurfaces observed between both counterparts.
In Table 1, the interaction energies, geometrical parameters (including interaction distances and angles), and values of the density at the BCP that connects the Hg atom with the electron-rich moiety are shown for a series of selected X-ray structures gathered from the PDB search.These were selected based on the X-ray resolution, the alkylated Hg moiety involved, and the interaction distance and angle values in order to provide a representative view of the interaction.As noted, the energy values obtained are far from a coordination bond energy (ranging between −10.0 and −1.9 kcal/mol) with Hg••• A distances comprised between 2.8 and 4.0 Å.
Energetic Study.To get further insights into the Hg SpBs present in these systems, we designed a computational study at the RI-MP2/def2-TZVP level of theory using a set of electronrich species and a MeHg molecule coordinated to a thiolate group (Figure 3) as theoretical models.The energetic results are shown in Table 2, and from their inspection, several interesting points arise.
First, in all cases, attractive interaction energies were obtained (between −23.1 and −0.9 kcal/mol), spread between weak (complex 8) and moderately strong values (complex 16).Also, the equilibrium distances obtained ranged between 2 and 3.5 Å, in line with the selected structures from the PDB search.As expected, those complexes involving Br − and Cl − (16 and 17) obtained the largest interaction energy values (−23.1 and −19.6 kcal/mol, respectively) in the study.In addition, complex 15 involving BF 4 − as the electron donor moiety achieved a less favorable interaction energy value due to its lower basicity compared to the monatomic anionic species.
Among the neutral complexes (1−14), complex 14 involving an indole ring obtained the most favorable SpB energy (−11.4kcal/mol) owing to the simultaneous establishment of an SpB and an HB with the MeHg molecule (see the QTAIM and NCIplot Analyses section).On the other hand, complex 8 involving the weakest Lewis base (OC) achieved the poorest interaction energy value of the set (−0.9 kcal/ mol).Among the π-system donors used (benzene and phenol), complex 10 involving the latter obtained a more favorable interaction energy value (−9.6 kcal/mol) due to (i) its higher π-basicity and (ii) the formation of an ancillary S•••HO HB vs a S•••CH HB in complex 1 (see Figure S1 for their respective QTAIM and NCIplot analyses).
Also, for the N-donating species (imidazole, HCN, NH 3 , and pyridine; complexes 5 to 7 and 11), complexes 6 and 11 involving imidazole and pyridine achieved the most favorable interaction energy values (−8.0 and −7.1 kcal/mol, respec-Table 3. Values of the Laplacian of ρ (∇ 2 ρ × 10 2 , in au), the Potential (V × 10 2 , in au) and Kinetic (G × 10 2 , in au) Energy Densities, and the Total Energy Density (H × 10 2 , in au) Gathered at the BCP That Characterizes the Hg SpB tively), although being weaker Lewis bases than NH 3 .This is due to the formation of ancillary HBs involving NH and CH groups from the imidazole and pyridine rings, respectively, and the thiolate group coordinated to MeHg (see Figure S1 for more details).On the other hand, complex 5 involving HCN as electron donor species obtained the lowest energy of this set (−4.2 kcal/mol), as expected.
Finally, among the O donor molecules (formaldehyde, formamide, dimethyl ether, and pyridine-N-oxide), complexes 3 and 12 involving formamide and pyridine-N-oxide obtained the largest interaction energy values (−7.9 and −7.2 kcal/mol, respectively).This was an unexpected result in the case of complex 3; however, a strong HB involving the NH 2 group of formamide and the S atom from the Hg moiety was undertaken, thus noticeably contributing to the total stabilization of this supramolecular complex.On the other hand, in complex 12 involving pyridine-N-oxide, this HB involved a CH group, being weaker than that in complex 3 (see the QTAIM and NCIplot Analyses section).
QTAIM and NCIplot Analyses.In Figure 4, the combined QTAIM and NCIplot analyses for some representative complexes are shown (the rest are included in Figure S1), and in all cases, a BCP (red sphere) and a bond path connecting the electron donor and Hg atoms were observed, which characterized the Spodium bonding interactions studied herein.Also, ancillary HBs (in complexes 3, 12, 14 and 15) and lp−π (complex 5) interactions were observed.For instance, in complexes 3, 12, and 14, the HBs involved the NH and CH groups from the formamide and indole moieties and the lone pairs of the S atom coordinated to the Hg center.On the other hand, in the case of complex 12, the lp−π interaction implied a lone pair from the S atom and the πsystem of the HCN molecule.Lastly, in complex 15, an ancillary HB interaction was described by the presence of a BCP connecting a F atom from the BF 4 − moiety and a CH group from the Hg coordination complex.Interestingly, the value of the density at the BCP that characterizes the SpB interaction exhibits a larger magnitude than that for the ancillary HB and lp−π interactions, thus highlighting the directing role of the Hg SpBs as the predominant noncovalent force in the supramolecular complexes studied herein (see Table 2 for the complete list of ρ × 10 2 values).
In Table 3, the values of the Laplacian at the BCP that characterize the Hg SpB (∇ 2 ρ × 100) are shown, resulting in positive values in all cases, as is common in closed shell calculations.Furthermore, the values of the potential (V × 100) and kinetic (G × 100) energy densities lie within the same range in all cases, thus confirming the noncovalent nature of the A•••Hg interaction (|Vr|/Gr) ≈ 1.
Regarding the NCIplot analyses, in all of the cases, a greenish isosurface was obtained between the electron donor molecule and the Hg moiety, thus indicating the presence of weak interactions.Also worth noting is the fact that the portion of the NCIplot isosurface devoted to the Hg SpB is bluish instead of greenish in the case of complexes 3, 7, and 15, thus indicating that the SpB interaction is noticeably stronger than the ancillary HBs, in line with the results obtained from QTAIM analyses.Finally, in the case of complexes 5 and 14, all surfaces exhibited a similar color, thus indicating a similar contribution of the ancillary HB and lp−π interactions and the Hg SpBs (see Figure S2 for the plots regarding the reduced density gradient (RDG) vs the sign(λ 2 )ρ).
NBO Analysis.To further investigate the participation of orbital contributions in the stabilization of the noncovalent complexes studied, we carried out NBO calculations focusing on the second-order perturbation analysis, which is useful to evaluate donor−acceptor interactions (see Table 4).Among the neutral complexes (1 to 14), the Hg SpBs were characterized by the interaction between either a lone pair  C−H orbital, and (iv) a bonding (BD) C−C orbital and an antibonding (BD*) C−H orbital.In all of these cases, the magnitude of the orbital interaction is lower than that observed for the Hg SpB, in line with the results derived from the QTAIM and NCIplot Analyses section (Figure 5).

■ CONCLUSIONS
In conclusion, we demonstrated the presence of Hg SpBs involving MeHg and EtHg in proteins using a combination of X-ray analysis and theoretical calculations at the RI-MP2/def2-TZVP level of theory.The PDB survey revealed a preference of the SpB at 4.0 and 110°as well as 4.25 and 130°, representing the distance (Hg•••A) and angle (X−Hg•••A), respectively.This is in alignment with the expected less hindered region in the X−Hg•••A plane.Besides, a variety of electron donor molecules was used to analyze the physical nature and extension in real space of the Hg SpB interaction (including O, S, and π-systems from aromatic residues) in a computational study.These computations were complemented with QTAIM and NCIplot analyses, which are utilized to further understand the weak nature of the interaction from a charge−density perspective as well as with NBO analyses, which highlighted the main orbital contributions responsible for the stabilization of the SpBs studied herein.We expect that the results derived from our study will be useful to those scientists devoted to protein engineering and bioinorganic chemistry as well as to expand the current knowledge of the SpBs among the chemical biology community.

Figure 1 .
Figure 1.(a) Distance distribution of Hg•••A contacts, (b) X−Hg−A angle distribution (X = C, S), and (c) radial distribution plot between Hg•••A distance and X−Hg•••A angle.The density scale is normalized with respect to the maximum count, where red represents maximum counts and blue represents minimum counts.

Figure 2 .
Figure 2. Spodium bonds (SpBs) in (a) 1IRK, (b) 5LU8, and (c) 3PYK structures.The interactions are magnified inside the square parts of the figure, also including QTAIM and NBO analyses of each SpB complex.In panel (d), the MEP surfaces of the MeHg and EtHg molecules are shown (energy values in kcal/mol at 0.001 au).Distances are measured as the shortest value between the Hg atom and the interacting AA.

Figure 3 .
Figure 3. Schematic representation of the compounds and complexes used in this study.

Figure 4 .
Figure 4. NCIplot analysis and QTAIM distribution of intermolecular bond critical points (BCP in red spheres) and bond paths in complexes 3, 5, 7, 12, 14, and 15.The value of the density at the BCPs characterizing the SpB interaction is also indicated in red.Ancillary interactions with their respective BCP density values are also included.NCIplot surfaces involve only intermolecular contacts between the Sp coordination complex and the electron donor molecule.NCIplot color range −0.04 au ≤ (signλ 2 )ρ ≤ + 0.04 au.Isosurface value RGD = 0.5 and ρ cutoff 0.04 au.
(LP) from an O, S, N, or F atom or a bonding (BD) C−C/N− C orbital belonging to the electron-rich species and an antibonding (BD*) Hg−C orbital from the MeHg moiety.The magnitude of these orbital interactions ranges from 0.19 kcal/ mol in complex 8 involving OC as an electron donor molecule to 4.88 kcal/mol in complex 6 involving an imidazole ring.On the other hand, in the case of the anionic complexes (15−17), the orbital contribution is larger in the case of the monatomic anions Cl − and Br − , with values of 18.30 and 16.90 kcal/mol, in line with their respective interaction energies.In addition, in complexes 1 to 3, 6, and 12 to 15, ancillary lp−π, HB, and CH−π interactions were also observed, involving (i) an LP from a S atom and an antibonding (BD*) C−C orbital, (ii) an LP from a S atom and an antibonding (BD*) C−H and N−H orbital, (iii) an LP from a F atom and an antibonding (BD*)

Figure
Figure S1 including additional QTAIM and NCIplot analyses and Figure S2 showing the NCI plots of reduced gradient vs sign(λ 2 )ρ and Cartesian coordinates of complexes 1−17 and of the PDB models used (PDF)

Table 1 .
List of PDB Codes Retrieved from the Search (PDB ID) Including the Interacting Partners (SpB Donor and AA), the Resolution of the Structure (in Å), the BSSE-Corrected Energies (ΔE BSSE , in kcal/mol), Geometrical Parameters (Distance d A•••Hg , in Å and A•••Hg−C Angle, in Degree), and the Value of the Density at the BCP That Characterizes the Hg SpBs (ρ × 10 2 ) at the RI-MP2/def2-TZVP Level of Theory a

Table 2 .
BSSE-Corrected Interaction Energies (ΔE BSSE , in kcal/mol), Geometrical Parameters (Distance D A•••Hg , in Å and A••• Hg−C Angle, in Degree), and the Value of the Density at the Bond Critical Point (ρ × 10 2 ) Involving the SpB and the Ancillary Interactions Present in Complexes 1−17 at the RI-MP2/def2-TZVP Level of Theory a a

Table 4 .
(2)or and Acceptor NBOs with an Indication of the Second-Order Interaction Energy E(2)in Complexes 1− 17 a BD, and BD* stand for lone pair, bonding orbital, and antibonding orbital, respectively.Energy values are in kcal/mol.
a LP,